Development of a TaqMan Allelic Discrimination assay for detection of single nucleotides polymorphisms associated with anti-malarial drug resistance

Abstract

Background: Anti-malarial drug resistance poses a threat to current global efforts towards control and elimination of malaria. Several methods are used in monitoring anti-malarial drug resistance. Molecular markers such as single nucleotide polymorphism (SNP) for example are increasingly being used to identify genetic mutations related to anti-malarial drug resistance. Several methods are currently being used in analysis of SNP associated with anti-malarial drug resistance and although each one of these methods has unique strengths and shortcoming, there is still need to improve and/or develop new methods that will close the gap found in the current methods.

Methods: TaqMan Allelic Discrimination assays for detection of SNPs associated with anti-malarial drug resistance were designed for analysis on Applied Biosystems PCR platform. These assays were designed by submitting SNP sequences associated with anti-malarial drug resistance to Applied Biosystems website. Eleven SNPs associated with resistance to anti-malarial drugs were selected and tested. The performance of each SNP assay was tested by creating plasmid DNAs carrying codons of interests and analysing them for analysis. To test the sensitivity and specificity of each SNP assay, 12 clinical samples were sequenced at codons of interest and used in the analysis. Plasmid DNAs were used to establish the Limit of Detection (LoD) for each assay.

Results: Data from genetic profiles of the Plasmodium falciparum laboratory strains and sequence data from 12 clinical samples was used as the reference method with which the performance of the SNP assays were compared to. The sensitivity and specificity of each SNP assay was establish at 100%. LoD for each assay was established at 2 GE, equivalent to less than 1 parasite/μL. SNP assays performed well in detecting mixed infection and analysis of clinical samples.

Conclusion: TaqMan Allelic Discrimination assay provides a good alternative tool in detection of SNPs associated with anti-malarial drug.

Figure 1

LoD for DHFR51 and DHFR108, MDR86 and MDR184, MDR1034 and MDR1042, DHPS581 SNP assays. Data showing LoD for allele1 and allele2 for each SNP assay where plasmid DNA carrying either allele1 or allele2 was used as a template. LoD for all the SNP assays was established at 2 GE. Data is shown indicating the SNP assay and the strain which the PCR fragment in the plasmid DNA was cloned from. Example 51-3D7 is data obtained from SNP assay DHFR51 using 16-3D7 plasmid DNA.

Figure 2

Performance of DHFR108, MDR1034 and DHPS581 SNP assays in mixed infection experiments. Plasmid DNAs carrying one of the alleles for each SNP assay were used in these experiments by mixing them in a each reaction. Plasmid DNAs were serially diluted 5-fold starting at 32000 GE down to 2 GE. In some experiments, the concentration of one of the plasmid DNA was kept constant while the other was serially diluted whereas in other experiments, both plasmid DNAs were serially diluted and mixed at equal DNA concentrations.

Figure 3

Manually called SNP assays. Data showing the manual call of MDR86 and MDR184 SNP assays. The automatic call for these SNP assays during these runs were undetermined however as the data shows, Allele1 and Allele2 ΔRn in these SNP assays were clearly separated. This data shows manual calls can be made with great confidence regardless of whether automatic calls are made or not. In addition, CT values showed a clear and distinct difference between the two alleles.

Figure 4

Allelic Discrimination plots for MDR86 and DHPS581 SNP assays. Two representative plots showing performance of two assays in analysis of clinical samples. These assays show clear separation between the signals derived from allele1 or allele2. Allele1 is shown in red and allele2 is shown in blue. Lime green represents a mixture of the two alleles (which was a positive control allele1/allele2 derived using a mixture of two plasmid DNAs).